关键词: 非编码小RNA, 消减杂交, 乳腺癌细胞

Abstract: The purpose of this paper is to build a new method that can detect the differences of small non-coding RNAs in breast cancer cells. This paper adopt the methods of isolating the total RNA from breast cancer cell lines MCF7 and nontumorigenic cell line HBL100, then adopting the RNA Isolation Kit to isolate the special size of RNAs（18-100 nt）, which were added poly（A）trails at the 3'-end and then reversing into the first strand of cDNA. After the subtractive hybridization of single stranded cDNA and RNAs, the differences of expressed small non-coding RNAs can be obtained with the method of using SA-PMPS Isolation System and the final result of hybridization can been tested by the internal reference of U6. Eventually, the products of hybridization were amplified through Nest PCR and PCR products were then lighted into T vector for sequencing. Results showed that after isolation, some special bands will be shown within 100 nt in the 10% polyacrylamide gel; the result of subtractive products PCR, which adopts the internal reference of U6, only shows visible band after 33 PCR circles. There were obvious bands within the size of 100 nt after Nest PCR and electrophoresis for the products of subtractive hybridization concluding the differences of expressed small non-coding RNAs were obtained. In this study, the improved subtractive hybridization can effectively identify the differences of expressed small non-coding RNAs in different cells.

Key words: Small non-coding, RNA, Subtractive hybridization, Breast cancer cell